Cd4 bv711

Left lungs from HD-Ad_RBD vaccinated and control mice were harvested and digested for 45 min at 37 °C in digestion buffer containing liberase 2 μg/ml and Type IV DNase I 25 units/ml. The lung infiltrated cells were cultured with purified RBD protein at 10 μg/ml for 12 h at 37 °C followed by a 6 h treatment with GolgiPlug (BD 555028). After blocking with FcγIII and FcγII receptors antibodies (BD Pharmingen, 553142), cells were stained with live/dead fixable cell stain (Invitrogen 34955), CD44 BV510, CD4 BV711, and CD8a APC-Cy™7 (BD) antibodies. Stained cells were fixed and permeabilized with Cytofix/Cytoperm Fixation/Permeabilization (BD 555028), and then intracellularly stained with anti-IFN-γ APC (BD). Cells were analysed on a Becton Dickinson LSR II CFI (SickKids Flow Cytometry Facility), using Flowjo × 10.0 software.

T-cell activation and proliferation were monitored using T-cell activation markers and a T-cell proliferation assay. In parallel to the main experiment, a sample of CellTraceTM Violet (CTV) (1:1000, cat. C34557, Thermo Fisher Scientific, USA) stained T cells (CD4 and CD8) from each donor was performed as per the protocol given by the manufacturer. At each time point a sample of cells was stained with CD69-PE-cy7 (1:200, cat. 557745, BD biosciences, USA), CD226-FITC (1:300, cat. 559788, BD Biosciences, USA) GLUT-1-(1:200, cat. 566580, BD Biosciences, USA) along with CD3-APCe780 (1:300, cat. 47-0032-82, eBioscience, USA), CD4-BV711 (1:400, cat. 563033, BD Bioscience, USA) and CD8-PerCP/Cy5.5 (1:200, cat. 344710, Biolegend, USA). Samples were analyzed using FACS to determine dynamic expression changes. The percentage of CTV- cells were analyzed to determine the T-cell proliferation rate at different time points.

Whole blood was used to calculate absolute leukocyte counts by Trucount analysis (BD Biosciences) according to manufacturer’s protocol. The following antibodies were used: CD45-PerCP (BioLegend), and CD3-APC-R700, CD4-BV711, CD8-BV786, all purchased from BD Biosciences. Using the bead count of the Trucount tube and the CD3⁺ cell count, absolute cell numbers of the proliferation/apoptosis and senescence panel were also calculated. In addition, for all individuals, HLA typing on whole blood was performed by flow cytometry at the first visit. For all these HLA-types, we characterized the known immunodominant CMV epitopes for which commercial dextramers are available. Antibodies used were HLA-A1/36-biotin/strep-PE-CF594, HLA-A2-V450, HLA-A3-APC, HLA-A24-PE, HLA-B7-FITC, and HLA-B8-PE-Cy7. Cells were measured on a Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo V10 software.

In vitro expanded cells were washed, counted, and plated in a 96 well plate at a density of 1 × 10⁶ cells/well. Cells were then stained with a mixture of the following antibodies: Fixable Viability Dye eFluor 506 (Thermo Fisher), CD3-AF700 (BD, RRID:AB_10597906), CD4-BV711 (BD, RRID:AB_2740432), and CD8-BV650 (Biolegend, RRID:AB_11125174) for 30 min at 4°C in the dark. Stained cells were then washed twice and resuspended in 100 μL PBS to be run on an LSR II flow cytometer (BD; a detailed protocol can be found at (https://doi.org/10.17504/protocols.io.bwu9pez6). FCS files produced from the LSR-II were then analyzed using FlowJo v10.8.2 software (Tree Star; RRID:SCR_008520; https://www.flowjo.com/solutions/flowjo).

Tumor cell suspensions (4 × 10⁶) prepared by mechanical and enzymatic dissociation^{16 (link)} were stained in 96-wells round bottom plates with live/dead staining (Blue fluorescent reactive dye, #L34962 Invitrogen) during 20 min at room temperature. For flow-cytometry analysis and sorting, Fc receptors were blocked with anti-FcR (anti-CD16 and CD32, at 5 µg/ml, Biolegend #101339). After two washes in PBS 2% FCS, cells were stained with the following antibodies (all used at 1:100): anti-CD11b-BV421 (#562605), CD64-APC (#558539), CD11c-PeCy7 (#557401), TCRβ-BV605 (#562840) and CD4-BV711 (#563050) all purchased from BD Pharmingen; anti-CD45-AF700 (#103128), Ly6C-APCCy7 (#128025), Ly6G-BV510 (#127633), F4/80 BV650 (#123149), CD206-PE (#141706), IA/IE-BV785 (#107645) all purchased from Biolegend, and CD8-PerCPef710 (#46-0081-82) purchased from eBioscience. After washing, cells were fixed in 1% PFA, stored at 4 °C, and acquired the next day on LSR II or FORTESSA (BD Bioscience). For detection of phosphorylated proteins, cell suspensions were stimulated 3 h with DMXAA 250 µg/ml, fixed immediately in PFA 4%, permeabilized with frozen methanol 90%, stained overnight with 1:100 pTBK1-PE (#13498) antibodies (purchased from Cell signaling), then washed and further stained for multicolor flow cytometry.